Novel Expression System for Combined Vaccine Production in Edwardsiella tarda Ghost and Cadaver Cells

To develop combined vaccine systems, we have generated Edwardsiella tarda ghosts (ETG) displaying a foreign protein on the outer membrane and also Ed. tarda cadaver (ETC) expressing a heterologous protein in the cytoplasm. Green fluorescent protein (GFP) was used as a model foreign protein. A constitutive promoter (EtPR C28-1) cloned newly from Ed. tarda was used as a promoter for the expression of foreign protein. Comparison of the strength of the new promoter with a commercially available constitutive promoter (P-HCE) showed higher expression levels of the novel expression system. The N-terminal domain of ice nucleation protein (InaN), an outer membrane protein of Pseudomonas syringae, was used as an anchor motif for surface display of GFP. By transformation of Ed. tarda with the constructed vectors, GFP was successfully expressed on the surface of ETG and in the cytoplasm of ETC. When compared to P-HCE driven expression, approximately more than 2 times of GFP was expressed on ETG and in ETC by EtPR C28-1 promoter when judged by fluorescent spectrophotometry. Furthermore, significantly higher expression of GFP on the surface of ETG by EtPR C28-1 than by P-HCE was demonstrated by serum agglutination assay. These results suggest that the newly cloned Ed. tarda constitutive promoter is capable to express foreign proteins not only on the surface of Ed. tarda ghosts but also in the cytoplasm of Ed. tarda cadavers, and can be used as an efficient promoter for the expression of heterologous antigens of the ETG and ETC-based combined vaccines.